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Gregor Matz, Bernhard Messerschmidt, and Herbert Gross, "Design and evaluation of new color-corrected rigid endomicroscopic high NA GRIN-objectives with a sub-micron resolution and large field of view," Opt. Express 24, 10987-11001 (2016)

Abstract

We demonstrate new GRIN-based endomicroscopic objectives for high resolution single photon fluorescence imaging modalities. Two endoscopic optical design approaches are presented in detail utilizing firstly diffractive and secondly refractive optical elements for the color correction in a spectral range from 488 nm to 550 nm. They are compared with their precursor device experimentally and by simulation. Inherent aberrations for off-axis field points could be lowered remarkably compared with the values of the state-of-the-art system by increasing the intrinsic optical complexity but maintaining the outer spatial dimensions. As a result, those presented objectives predict a diffraction-limited imaging of objects up to 300 μm in diameter with a numerical aperture of 0.8 while keeping an overall outer diameter of the assembly at 1.4 mm. Lastly, confocal fluorescence imaging experiments focus on the comparison between the numerical predicted and the practically achieved quality parameters.

Fig. 3 Evaluation of the influence of the undesired diffraction orders of the DOE on the imaging performance for confocal single photon fluorescence imaging processes; The calculation is performed by convolving the PSF in the object plane (λ =488 nm) with the PSF in the image plane (λ =550 nm) while assuming a complete conversion of the energy at the specimen; The table on the left presents the ratio between the detected (EDetected) and emitted amount of energy (EEmitted) passing through the on-axis pinhole in the image plane of the size of one airy diameter (2.2 μm; @λ =550 nm); the DOE is assumed to work perfectly in either the zeroth, first or second diffraction order for the illumination and detection for case (a); the DOE is assumed to work perfectly in the first order for the illumination and perfectly in either the zeroth, first or second diffraction order for the detection in case (b) (η0/1/2 is the diffraction efficiency of the corresponding order).

Fig. 5 Monochromatic Strehl ratio of the on-axis (a) and 100 μm (object-sided) off-axis (b) field point as a function of the object-sided working distance and the wavelength evaluated for the precursor (left), DiPolyC (middle) and RePolyC (right), respectively; the black lines confine the region with a Strehl ratio larger than 0.8 and thus predict a diffraction limited imaging performance; the improved off-axis correction for the recent developments as well as a good spherochromatic and axial chromatic correction for the targeted wavelengths of 488 nm and 550 nm is obvious; RePolyC shows additionally a finite on-axis and off-axis axial chromatic aberration for the extended spectral range.

Fig. 7 Desired, experimentally determined wavefront at 633 nm of the GRIN-lens measured with a shearing interferometer (left); Remaining undesired higher order aberrations of the same wavefront after subtracting a Zernike fringe fit up to 4th order; transverse axes normalized on unity and W scaled in wavelengths (@ 633 nm).

Fig. 8 Experimental setup for the evaluation of the endoscopic GRIN-objectives; the object imaged by the endoscopic system was scanned and digitalized confocally by the use of a laser scanning microscope; a self build x-y-z micrometer positing system enabled a precise positioning of the two objectives to each other.

Fig. 9 (a) Confocal images of subresolution beads (FluoSpheres Carboxylate 0.2 μm yellow-green 505/515, 2% solids, diluted 1:10.000 in water) observed through RePolyC (left), DiPolyC (middle) and their precursor device (right) for progressively increased image zones (top to bottom); Excitation at 488 nm and detection from 505 nm to 540 nm with an adapted laser intensity for every measurement; The increased performance for RePolyC and DiPolyC in comparison with the precursor is obvious; Scale bar corresponds to 2 μm in image plane (b) confocal z-stack of a subresolution sphere in fluorescence mode with DiPolyC at a lateral distance of 50 μm from the optical axis in the image plane; Scale bar corresponds to 2 μm in image plane (c) Determination of the corresponding axial FWHM in the object plane by a Gaussian fit to the intensity maximums IMax(z) of the lateral Gaussian fits.

Fig. 10 Experimentally measured lateral (left) and axial (right) resolution of RePolyC (green), DiPolyC (blue) as well as their precursor (red) depending on the radial size of the corresponding object zone; the later FWHM values are converted to the object plane by dividing by the lateral magnification; the axial FWHM’s are measured in the object plane directly since the MO is physically connected with the endomicroscopic objective.

Fig. 11 Analysis of tangential (T) and sagittal (S) resolution (black lines) and the axial peak-intensities (red lines) for every slice of the z-stack by evaluating the PSF at an image height of 369 μm; the FWHM values are converted to the object plane by making use of the magnification; determination of the lateral FWHM’s at the circle of least confusion by averaging the values for the tangential and sagittal plane confined by the blue bar (compare Fig. 10).

Fig. 12 Confocal images of a 1.76±0.16 μm thick fluorescine layer measured with DiPolyC(middle), RePolyC (botton) and their precursor (top) at selective axial distances of the image side (left) and the respective highest intensity projections (right side); the images are scaled in the image plane of the GRIN-systems whereas the red circles symbolize the margin of the back surface of the corresponding GRIN-system with a diameter of 1 mm; excitation light: 488 nm; MO: UplanSApo10x0.4 NA; white scale bar corresponds to 100 μm in object plane, respectively.

Fig. 13 Experimentally measured confocal PSF intensity response averaged over four samples of a 1.76±0.16 μm thick fluorescine layer for RePolyC (green), DiPolyC (blue) and its precursor (red) (left); Predicted PSF intensity response when all surfaces would be anti reflection-coated and the NA of the MO would be matched with the NA of the GRIN-objective at the intermediate image plane (right); The radial distance is converted to the object plane by using the lateral magnification of the corresponding probe.